Colorectal Cancer

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.

Adverse events of upper GI endoscopy

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Biochemical and genetic findings accumulated over the past decade have established that the condensation of eukaryotic DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. This genetic repression extends from the level of the nucleosome, the primary unit of chromatin organization, where coiling of DNA on the surface of the nucleosome core particle impedes access to the transcriptional apparatus, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (for reviews see Refs. 1van Holde K. Zlatanova J. Arents G. Moudrianakis E. Elgin S.C.R. Chromatin Structure and Gene Expression. Oxford University Press, Oxford, UK1995: 1-26Google Scholar, 2Ramakrishnan V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (132) Google Scholar, 3Pruss D. Hayes J.J. Wolffe A.P. Bioessays. 1995; 17: 161-170Crossref PubMed Google Scholar, 4Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Crossref PubMed Google Scholar, 5Kornberg R.D. Lorch Y. Annu. Rev. Cell Biol. 1992; 8: 563-589Crossref PubMed Google Scholar, 6Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 149-188Crossref PubMed Google Scholar and 105Koshland D. Strunnikov A. Annu. Rev. Cell Biol. 1996; 12: 305-333Crossref Scopus (283) Google Scholar). Chromatin structure is inextricably linked to transcriptional regulation, and recent studies show how chromatin is perturbed so as to facilitate transcription (for reviews see Refs. 7Adams C.C. Workman J.L. Cell. 1993; 72: 305-308Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 8Paranjape S.M. Kamakaka R.T. Kadonaga J.T. Annu. Rev. Biochem. 1994; 63: 265-297Crossref PubMed Google Scholar, 9Kornberg R.D. Lorch Y. Curr. Opin. Cell Biol. 1995; 7: 371-375Crossref PubMed Scopus (95) Google Scholar, 10Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 12Krude T. Elgin S.C.R. Curr. Biol. 1996; 6: 511-515Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we review the substantial advances in the identification of histone acetyltransferases and histone deacetylases, whose opposing activities establish the steady-state level of histone acetylation, and progress in studies of multicomponent systems that require energy for the process of nucleosome disruption.Histone AcetylationSince the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar).Tetrahymena Histone Acetyltransferase A and Yeast Gcn5In a convergence of biochemical and genetic studies, cloning of the p55 catalytic subunit of Tetrahymena nuclear (A-type) histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; P/CAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element binding protein; SAS, something about silencing; MOZ, monocytic leukemia zinc finger; MOF, males absent on the first; TAF, TBP-associated factor; TBP, TATA-binding protein; HDAC, histone deacetylase. revealed substantial sequence identity with yeast Gcn5, previously defined genetically as a transcriptional coactivator (22Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1210) Google Scholar). The catalytic domain of the Gcn5 HAT is required for coactivator function in vivo, providing a genetic link between histone modification and transcriptional activation (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar). As a human GCN5 homolog has been identified, this HAT is likely to be widely conserved (24Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (154) Google Scholar, 25Wang L. Mizzen C. Ying C. Candau R. Barlev N. Brownell J. Allis C.D. Berger S.L. Mol. Cell. Biol. 1997; 17: 519-527Crossref PubMed Google Scholar). Bacterially expressed yeast Gcn5 protein acetylates free histone H3 strongly at lysine 14 and histone H4 weakly at lysines 8 and 16 (26Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (480) Google Scholar). However, unlike the native HAT A enzyme, recombinant Gcn5 cannot acetylate nucleosomal histones, implying that other subunits in the complex must influence its activity on chromatin. Genetic and biochemical studies reveal at least two interacting proteins, Ada2 and Ada3, that form a complex with Gcn5 (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar,27Marcus G.A. Silverman N. Berger S.L. Horiuchi J. Guarente L. EMBO J. 1994; 13: 4807-4815Crossref PubMed Scopus (234) Google Scholar, 28Horiuchi J. Silverman N. Marcus G.A. Guarente L. Mol. Cell. Biol. 1995; 15: 1203-1209Crossref PubMed Google Scholar, 29Georgakopoulos T. Gounalaki N. Thireos G. Mol. Gen. Genet. 1995; 246: 723-728Crossref PubMed Scopus (53) Google Scholar). Binding of Ada2 to the activation domains of the transcriptional activators VP16 and Gcn4 in vitro suggests a mechanism by which promoter targeting of Gcn5 might be achieved (30Silverman N. Agapite J. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11665-11668Crossref PubMed Scopus (96) Google Scholar,31Barlev N.A. Candau R. Wang L. Darpino P. Silverman N. Berger S.L. J. Biol. Chem. 1995; 270: 19337-19344Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar).P/CAF, p300/CBP, SAS, MOZ, and MOFAn increasing number of putative or demonstrated histone acetyltransferases have emerged in the past year. P/CAF (p300/CBP-associated factor) is a novel histone acetyltransferase isolated on the basis of sequence similarity to human and yeast Gcn5 (32Yang X.-J. Ogryzko V.V. Nishizawa J. Howard B.H. Nakayani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1283) Google Scholar), which interacts with the highly related transcriptional coactivators p300/CBP. Like Gcn5, recombinant P/CAF has intrinsic HAT activity for free histones H3 and H4, but unlike Gcn5, P/CAF is also able to acetylate nucleosomal histone H3. p300/CBP (CREB-binding protein) is itself a histone acetyltransferase with no resemblance in sequence to the other acetyltransferases (33Ogryzko V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2274) Google Scholar, 106Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1480) Google Scholar). The bacterially expressed p300/CBP protein is unique among HAT polypeptides in that it can acetylate all four core histones free in solution or when complexed in the nucleosome; acetylation on histone H4 occurs at lysines 5, 8, 12, and 16, the same positions that are subject to acetylation in vivo (32Yang X.-J. Ogryzko V.V. Nishizawa J. Howard B.H. Nakayani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1283) Google Scholar). p300 and CBP are known to physically interact with numerous transcription factors activated by signaling cascades, including CREB, c-Jun/v-Jun, Fos, and nuclear hormone receptors, and are also targets for the E1A oncoprotein (for a review see Ref. 34Janknecht R. Hunter T. Nature. 1996; 383: 22-23Crossref PubMed Scopus (337) Google Scholar). Whether histones are substrates of p300 in vivo remains to be determined.The yeast SAS, human MOZ and Tip60, and fly MOF proteins constitute a different class of putative acetyltransferases, characterized by a ∼300-amino acid region of significant similarity that contains a C2CH zinc finger motif and a subregion similar to HATs and other acetyltransferases (35Reifsnyder C. Lowell J. Clarke A. Pillus L. Nat. Genet. 1996; 14: 42-49Crossref PubMed Scopus (234) Google Scholar, 36Borrow J. Stanton Jr., V.P. Andresen J.M. Becher R. Behm F.G. Chaganti R.S.K. Civin C.I. Disteche C. Dube I. Frischauf A.M. Horsman D. Mitelman F. Volinia S. Watmore A.E. Housman D.E. Nat. Genet. 1996; 14: 33-41Crossref PubMed Scopus (609) Google Scholar, 37Kamine J. Elangovan B. Subramanian T. Coleman D. Chjnnadurai G. Virology. 1996; 216: 357-366Crossref PubMed Scopus (237) Google Scholar, 38Hilfiker A. Hilfiker-Kleiner D. Pannuti A. Lucchesi J. EMBO J. 1997; 16: 2054-2060Crossref PubMed Scopus (353) Google Scholar). The biochemical properties or substrate specificities of these proteins involved in silencing (SAS), transcriptional activation (Tip60), leukemogenesis (MOZ), and dosage compensation (MOF) have not yet been described. Interestingly, recurrent translocation in a subtype of acute myeloid leukemia generates a novel fusion of MOZ with CBP, suggesting that the aberrant acetylation of histones or other chromosomal proteins could mediate leukemogenesis (36Borrow J. Stanton Jr., V.P. Andresen J.M. Becher R. Behm F.G. Chaganti R.S.K. Civin C.I. Disteche C. Dube I. Frischauf A.M. Horsman D. Mitelman F. Volinia S. Watmore A.E. Housman D.E. Nat. Genet. 1996; 14: 33-41Crossref PubMed Scopus (609) Google Scholar).TAF250TFIID, the general transcription factor complex of TBP (TATA-binding protein) and the associated TAF proteins, has been found to contain a HAT activity. Unique among the various TAFs, TAFII250 alone or in the TFIID complex has both a serine kinase activity selective for RAP74 (a subunit of TFIIF) and a HAT activity (39Dikstein R. Ruppert S. Tjian R. Cell. 1996; 84: 781-790Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 40Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J.L. Berger S.L. Kouzarides T. Nakatani Y. Aliis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Like Gcn5, TAFII250 preferentially acetylates free histone H3 over H4 and has little or no activity on nucleosomal histones (40Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J.L. Berger S.L. Kouzarides T. Nakatani Y. Aliis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). The domain of TAFII250 responsible for HAT activity maps to the central, conserved region and shows no obvious sequence similarity to other HATs. The finding of HAT activity in TAFII250 implies that TFIID could contribute toward destabilizing nucleosomes over core promoter elements, although the physiological substrates of the HAT activity remain to be determined. TFIID may possess yet an another capacity for assisting nucleosome disorder. Portions of several TAFs (DrosophilaTAFII42 and TAFII62) adopt a histone octamer-like substructure that might be employed as a histone octamer-like substructure, which could serve as a competitor for DNA binding with the core histones (41Hoffman A. Chiang C.-M. Oelgeschlager T. Xie X. Burley S.K. Nakatani Y. Roeder R. Nature. 1996; 380: 356-359Crossref PubMed Scopus (157) Google Scholar, 42Nakatani Y. Bagby S. Ikura M. J . Biol. Chem. 1996; 271: 6575-6578Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 43Xie X. Kokubo T. Cohen S.L. Mirza U.A. Hoffman A. Chait B.T. Roeder R. Nakatani Y. Burley S.K. Nature. 1996; 380: 316-322Crossref PubMed Scopus (223) Google Scholar).Histone DeacetylasesIn parallel with the identification of HAT enzymes, studies of histone deacetylase (HDAC) enzymes in human, yeast, andDrosophila have also advanced significantly. Affinity chromatography with the ligand trapoxin, a high affinity, irreversible inhibitor, resulted in the purification and cloning of a human deacetylase (44Tauton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1456) Google Scholar) composed of a catalytic subunit, HD-1, renamed HDAC1 (45Hassig C.A. Fleischer T.C. Billin A.N. Schreibert S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar), and a tightly associated WD repeat protein, RbAp48. The sequence of the HDAC1 showed very strong sequence identity to yeast Rpd3, previously identified genetically to be necessary for full repression and activation of a subset of genes (46Vidal M. Gaber R. Mol. Cell. Biol. 1991; 11: 6317-6327Crossref PubMed Google Scholar). There are five members of theRPD3 family in yeast, two of which (HDA1 andRPD3) are components of the major histone deacetylase activities, which fractionate as 350-kDa (HDA) and 600-kDa (HDB) complexes (47Carmen A.A. Rundlett S.E. Grunstein M. J. Biol. Chem. 1996; 271: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 48Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (495) Google Scholar).As anticipated from the catalytic properties of the encoded proteins, deletions of the HDA1 and RPD3 genes strongly reduce HDA and HDB activities, leading to hyperacetylation of histones H3 and H4 in vivo. However, the phenotypes of these deletions are somewhat surprising, as they increase repression rather than increase activation of telomeric loci (48Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (495) Google Scholar). Similarly, mutation of a Drosophila homolog of RPD3 displays an enhancer of position effect variegation phenotype, suggesting that loss of wild-type Drosophila RPD3 function as a histone deacetylase leads to increased gene silencing (49De Rubertis F. Kadosh D. Henchoz S. Pauli D. Reuter G. Struhl K. Spierer P. Nature. 1996; 384: 589-591Crossref PubMed Scopus (190) Google Scholar). These findings may perhaps be related to the acetylation of histone H4 at lysine 12, which is required for transcriptional silencing in yeast (50Braunstein M. Sorbel R.E. Allis C.D. Turner B.M. Broach J.R. Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Google Scholar) and which is also associated with heterochromatin in Drosophila (51Turner B.M. Birley A.J. Lavender J. Cell. 1992; 69: 375-384Abstract Full Text PDF PubMed Google Scholar), despite net hypoacetylation.More in line with the anticipated involvement of histone deacetylases in transcriptional repression is the physical association of the heteromeric Mad (Mxi1)/Max DNA binding repressors involved in controlling cell proliferation and differentiation with mammalian RPD3 homologs and with Sin3, a conserved transcriptional co-repressor genetically linked to yeast Rpd3 (45Hassig C.A. Fleischer T.C. Billin A.N. Schreibert S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 52Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar). Immunopurification studies also reveal additional, novel polypeptides associated with Sin3 and histone deacetylase in human cell extracts (54Zhang Y. Iranti R. Erdjument-Bromage H. Tempst P. Reinberg D. Cell. 1997; 89: 357-364Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). YY1, a mammalian transcription factor that can serve as a repressor to histone deacetylase W.M. C. Y. D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar), and and for nuclear hormone receptors, interact with complexes mammalian Sin3 and histone deacetylase L. R. H. J. L. N. D. Nature. 1997; PubMed Google Scholar, T. T.M. M. 1997; PubMed Google Scholar, L. D. Hassig C.A. Ayer D.E. Schreiber S.L. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). of genes by the yeast protein also of a complex Sin3 and Rpd3 D. Struhl K. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). This of strong for a of repression by the of histone deacetylase complexes to the of so of chromatin structure a of that for is the of nucleosomal histones during the histones or from and how might a structure interactions between DNA binding S. M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google nucleosomes that are or to the structure of DNA have different for in chromatin are by proteins as and Burley S.K. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and by the complex itself L. A. D. M. Cell. 1997; 89: Full Text Full Text PDF PubMed Google chromatin are for a gene and at which of the activation the binding of regulators, of the or activities to specific chromosomal or they the chromatin of for other enzymes are by is the of the modification a nucleosome and how is the modification the activities of the histone acetyltransferases and histone deacetylases with the family of nucleosome on higher order chromatin or is it by higher order chromatin to these and other not only for the of gene but also for of Biochemical and genetic findings accumulated over the past decade have established that the condensation of eukaryotic DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. This genetic repression extends from the level of the nucleosome, the primary unit of chromatin organization, where coiling of DNA on the surface of the nucleosome core particle impedes access to the transcriptional apparatus, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (for reviews see Refs. 1van Holde K. Zlatanova J. Arents G. Moudrianakis E. Elgin S.C.R. Chromatin Structure and Gene Expression. Oxford University Press, Oxford, UK1995: 1-26Google Scholar, 2Ramakrishnan V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (132) Google Scholar, 3Pruss D. Hayes J.J. Wolffe A.P. Bioessays. 1995; 17: 161-170Crossref PubMed Google Scholar, 4Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Crossref PubMed Google Scholar, 5Kornberg R.D. Lorch Y. Annu. Rev. Cell Biol. 1992; 8: 563-589Crossref PubMed Google Scholar, 6Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 149-188Crossref PubMed Google Scholar and 105Koshland D. Strunnikov A. Annu. Rev. Cell Biol. 1996; 12: 305-333Crossref Scopus (283) Google Scholar). Chromatin structure is inextricably linked to transcriptional regulation, and recent studies show how chromatin is perturbed so as to facilitate transcription (for reviews see Refs. 7Adams C.C. Workman J.L. Cell. 1993; 72: 305-308Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 8Paranjape S.M. Kamakaka R.T. Kadonaga J.T. Annu. Rev. Biochem. 1994; 63: 265-297Crossref PubMed Google Scholar, 9Kornberg R.D. Lorch Y. Curr. Opin. Cell Biol. 1995; 7: 371-375Crossref PubMed Scopus (95) Google Scholar, 10Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 12Krude T. Elgin S.C.R. Curr. Biol. 1996; 6: 511-515Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we review the substantial advances in the identification of histone acetyltransferases and histone deacetylases, whose opposing activities establish the steady-state level of histone acetylation, and progress in studies of multicomponent systems that require energy for the process of nucleosome Histone AcetylationSince the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar). the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar). Tetrahymena Histone Acetyltransferase A and Yeast Gcn5In a convergence of biochemical and genetic studies, cloning of the p55 catalytic subunit of Tetrahymena nuclear (A-type) histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; P/CAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element binding protein; SAS, something about silencing; MOZ, monocytic leukemia zinc finger; MOF, males absent on the first; TAF, TBP-associated factor; TBP, TATA-binding protein; HDAC, histone deacetylase. revealed substantial sequence identity with yeast Gcn5, previously defined genetically as a transcriptional coactivator (22Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1210) Google Scholar). The catalytic domain of the Gcn5 HAT is required for coactivator function in vivo, providing a genetic link between histone modification and transcriptional activation (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar). As a human GCN5 homolog has been identified, this HAT is likely to be widely conserved (24Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (154) Google Scholar, 25Wang L. Mizzen C. Ying C. Candau R. Barlev N. Brownell J. Allis C.D. Berger S.L. Mol. Cell. Biol. 1997; 17: 519-527Crossref PubMed Google Scholar). Bacterially expressed yeast Gcn5 protein acetylates free histone H3 strongly at lysine 14 and histone H4 weakly at lysines 8 and 16 (26Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (480) Google Scholar). However, unlike the native HAT A enzyme, recombinant Gcn5 cannot acetylate nucleosomal histones, implying that other subunits in the complex must influence its activity on chromatin. Genetic and biochemical studies reveal at least two interacting proteins, Ada2 and Ada3, that form a

The role of endoscopy in the management of choledocholithiasis

REVIEW ARTICLES: Platelet G protein-coupled receptors in hemostasis and thrombosis

Structure and function of the ventricular tachycardia isthmus.

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.

The genetic instabilities of (CCTG.CAGG)(n) tetranucleotide repeats were investigated to evaluate the molecular mechanisms responsible for the massive expansions found in myotonic dystrophy type 2 (DM2) patients. DM2 is caused by an expansion of the repeat from the normal allele of 26 to as many as 11,000 repeats. Genetic expansions and deletions were monitored in an African green monkey kidney cell culture system (COS-7 cells) as a function of the length (30, 114, or 200 repeats), orientation, or proximity of the repeat tracts to the origin (SV40) of replication. As found for CTG.CAG repeats related to DM1, the instabilities were greater for the longer tetranucleotide repeat tracts. Also, the expansions and deletions predominated when cloned in orientation II (CAGG on the leading strand template) rather than I and when cloned proximal rather than distal to the replication origin. Biochemical studies on synthetic d(CAGG)(26) and d(CCTG)(26) as models of unpaired regions of the replication fork revealed that d(CAGG)(26) has a marked propensity to adopt a defined base paired hairpin structure, whereas the complementary d(CCTG)(26) lacks this capacity. The effect of orientation described above differs from all previous results with three triplet repeat sequences (including CTG.CAG), which are also involved in the etiologies of other hereditary neurological diseases. However, similar to the triplet repeat sequences, the ability of one of the two strands to form a more stable folded structure, in our case the CAGG strand, explains this unorthodox "reversed" behavior.

Disruption of Performance in the Five-Choice Serial Reaction Time Task Induced By Administration of N-Methyl-D-Aspartate Receptor Antagonists: Relevance to Cognitive Dysfunction in Schizophrenia

Part 8: Advanced life support: 2010 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations

The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae. The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work. Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP. Protein kinase A phosphorylated Opi1p on multiple serine residues. The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser31 and Ser251, respectively, within Opi1p were substrates for protein kinase A. Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins. In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein. The physiological consequence of the phosphorylation of Opi1p at Ser31 and Ser251 was examined by measuring the effects of the S31A and S251A mutations on the expression of the UASINO-containing gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1Delta mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p. These data supported the conclusion that phosphorylation of Opi1p at Ser31 and Ser251 mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene.

The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis "two-component" histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.

The K+-Cl- cotransporter (KCC) isoforms constitute a functionally heterogeneous group of ion carriers. Emerging evidence suggests that the C terminus (Ct) of these proteins is important in conveying isoform-specific traits and that it may harbor interacting sites for 4beta-phorbol 12-myristate 13-acetate (PMA)-induced effectors. In this study, we have generated KCC2-KCC4 chimeras to identify key functional domains in the Ct of these carriers and single point mutations to determine whether canonical protein kinase C sites underlie KCC2-specific behaviors. Functional characterization of wild-type (wt) and mutant carriers in Xenopus laevis oocytes showed for the first time that the KCCs do not exhibit similar sensitivities to changes in osmolality and that this distinguishing feature as well as differences in transport activity under both hypotonic and isotonic conditions are in part determined by the residue composition of the distal Ct. At the same time, several mutations in this domain and in the proximal Ct of the KCCs were found to generate allosteric-like effects, suggesting that the regions analyzed are important in defining conformational ensembles and that isoform-specific structural configurations could thus account for variant functional traits as well. Characterization of the other mutants in this work showed that KCC2 is not inhibited by PMA through phosphorylation of its canonical protein kinase C sites. Intriguingly, however, the substitutions N728S and S940A were seen to alter the PMA effect paradoxically, suggesting again that allosteric changes in the Ct are important determinants of transport activity and, furthermore, that the structural configuration of this domain can convey specific functional traits by defining the accessibility of cotransporter sites to regulatory intermediates such as PMA-induced effectors.

Section 13: Evaluation and Therapy for Heart Failure in the Setting of Ischemic Heart Disease